PROJECT SUMMARY Breathing is vital for survival, and failure to breathe is fatal. This has become tragically evident in the context of the current opioid crisis. Breathing disturbances are also the cause of sleep apnea, which is another health issue of epidemic proportions. At the core of all these disturbances are neuronal networks located within the brainstem. Two of these networks, the preBtzinger complex (preBtC) and the parafacial respiratory group (pFRG) are thought to give rise to inspiration and active expiration, respectively. During the initial funding period of this grant, we identified a third excitatory microcircuit, the postinspiratory complex (PiCo), which gives rise to a third breathing phase: postinspiration ? the expiratory phase that follows inspiration. Based on our discovery, we proposed the triple oscillator hypothesis: i.e. three excitatory microcircuits (preBtC, pFRG, PiCo) give rise to the three phases of breathing. However, the discovery of PiCo raised an important, unresolved issue: what is the role of the so-called Btzinger complex (BtC), a fourth region that contains respiratory neurons, and that is located rostral of the preBtC? Here we test the overarching hypothesis that the preBtC is not a small microcircuit, as previously thought, but that this network forms a dynamically regulated column contiguous with the BtC. The extent of this column is dynamically regulated by synaptic inhibition, chemo- and mechanosensory afferents. The project tests this hypothesis in three specific aims: Aim 1 maps the extent of respiratory activity along the medullary column. We will use electrophysiological, calcium imaging and optogenetic approaches to characterize the neuronal discharge patterns within this column. Aim 2 investigates the cellular determinants that control the extent of this column using intracellular and optogenetic recordings. We specifically test the hypothesis that a balance between synaptic inhibition, and excitation regulates the regularity, frequency and spatial extent of the column. To conduct aims 1 and 2 we will employ horizontal brainstem slices that isolate the entire ventral medulla and that are amenable to a rigorous cellular and network analysis. Aim 3 explores the dynamic regulation of the column in alert and anesthetized in vivo animals. We test the hypothesis that vagal and chemosensory afferents play a critical role in regulating the spatial extent of this column by activating inhibitory neurons that are capable of shrinking and extending the inspiratory rhythmogenic network. The proposed research may lead to a better understanding of the fundamental question: how the brain generates rhythmic motor activity and how it integrates sensory information. Insights gained will also have important implications for understanding the cellular and systems level mechanisms underlying the mortality and morbidity associated with breathing disorders.